JP6599583B1 - Multigene disrupted Aspergillus microorganism and method for producing the same - Google Patents

Abstract

Disclosed is a transformed Aspergillus microorganism lacking a selectable marker gene that can be used in at least two types of marker recycling methods, and a composition for use thereof. SOLUTION: A transformed Aspergillus microorganism lacking a selectable marker gene that can be used for at least two types of marker recycling methods on a chromosome, and a loop-out region and a marker recycling method between homologous recombination regions A composition for transforming Aspergillus microorganisms comprising at least two kinds of nucleic acid fragments containing a selectable marker gene that can be used in the above, wherein the selectable marker gene is a tryptophan biosynthetic gene and a gene different from the tryptophan biosynthetic gene Including. [Selection] Figure 11

Description

The present invention relates to a transformed Aspergillus microorganism in which at least two genes on the chromosome are disrupted and a method for producing the same.

Aspergillus ( Aspergillus ) microorganisms are used as a koji mold used in the koji making process, which is one of the processes for producing fermented foods such as soy sauce and miso, and are also used for various substance production. Therefore, there is a need for a technique for modifying Aspergillus microorganisms so as to express a desired trait by, for example, destroying a target gene on a chromosome.

As a method for destroying a target gene on a host chromosome, there is a marker recycling method (also called a marker recycling method). In the marker recycling method, after a target gene is replaced with a selection marker gene by homologous recombination, the selection marker gene is removed so that the selection marker gene does not remain on the chromosome of the transformant, and the selection marker gene is regenerated. It is a method of using.

The selection marker gene used in the marker recycling method is required to have a system that can easily confirm the replacement by the selection marker gene and the removal of the selection marker gene. As a selection marker gene that can be used in such a marker recycling method, the pyrG gene is used in Aspergillus microorganisms.

Aspergillus microorganisms can grow without the addition of uridine and / or uracil (hereinafter sometimes referred to as uridine / uracil) by having the pyrG gene on the chromosome, but biosynthesis of uridine monophosphate 5-fluoroorotic acid (5-FOA), which is an analog of orotidine 5'-monophosphate, which is an intermediate of the above, is synthesized to toxic 5-fluorouracil (5-FU). Therefore, an Aspergillus microorganism having a pyrG gene on a chromosome cannot grow even if cultured in the presence of 5-FOA. In contrast, since Aspergillus microorganisms that do not have a pyrG gene on their chromosomes cannot metabolize 5-FOA, they can grow even in the presence of 5-FOA, but are uridine / uracil-requiring.

As a selectable marker gene that can be used in the marker recycling method, such as the pyrG gene, counter selection that makes it impossible to grow in an environment containing a specific drug (such as 5-FOA) by expressing the gene. It is required that the gene be a selectable marker gene. In other words, unless a drug that can be used for counter selection is found, the selectable marker gene cannot be used in the marker recycling method.

Using this property, a transformant in which the target gene on the chromosome of the Aspergillus microorganism lacking the pyrG gene is replaced with the pyrG gene is selected using a medium not containing uridine / uracil, and then the selected transformant is selected as 5- By performing counter selection using a FOA-containing medium, a transformant lacking the target gene and the pyrG gene can be obtained as a 5-FOA resistant strain.

However, at present, the above-described pyrG gene is only used as a selectable marker gene used in the marker recycling method for Aspergillus microorganisms. In addition, in the counter selection of the niaD-deficient strain using the drug and the counter selection of the sC-deficient strain, since the selective pressure of the drug is weak, the background growth of the non-deficient strain is observed. Currently, it is rarely used in the marker recycling law.

On the other hand, Patent Document 1 describes the presence or absence of a trpC gene, which is a gene involved in tryptophan biosynthesis, in a transformant using Rhizopus delmar as a host. ) Is evaluated by confirming the presence or absence of growth in the presence. Non-Patent Document 1 describes transformed Aspergillus acreitas lacking the pyrG gene and the argB gene, which is a biosynthetic gene for arginine, as selection marker genes.

WO2017 / 135317 pamphlet

TANI et al., AMB Express, 2013, 3: 4, https://amb-express.springeropen.com/articles/10.1186/2191-0855-3-4

In Patent Document 1, trpC gene-deficient strains are selected based on the presence or absence of growth in the presence of 5-FAA. However, Patent Document 1 does not describe that the trpC gene can be used as a recyclable selection marker gene for use in the marker recycling method.

In addition, in the transformed Aspergillus acreata described in Non-Patent Document 1, the missing argB gene is not a selectable marker gene that can be used in the marker recycling method. Further, in Non-Patent Document 1, using another selectable marker gene instead of the argB gene, for example, using a specific selectable marker gene among genes involved in biosynthesis of many amino acids, There is no description.

Furthermore, when the host organism Aspergillus genus microorganism is transformed by the marker recycling method, it can be recycled to the same extent as the pyrG gene, and a selection marker gene that can be used in place of the pyrG gene has been known to date. Not. In particular, a system that can be used together with the pyrG gene and that can be easily confirmed for replacement and removal by a selectable marker gene that can be used in the marker recycling method is hardly known so far.

Accordingly, a first problem to be solved by the present invention is to provide a transformed Aspergillus genus microorganism lacking at least two types of selectable marker genes that can be used in the marker recycling method and a method for producing the same.

The second problem to be solved by the present invention is to use at least a composition comprising at least two nucleic acid fragments containing a selectable marker gene, using a marker recycling method, and at least present on the chromosome of a transformed Aspergillus microorganism. It is to provide a method for deleting two types of target genes.

As a result of intensive studies to solve the above problems, the present inventor has come to focus on genes involved in tryptophan biosynthesis among various genes that can be used. Then, using the transformed Aspergillus microorganism lacking the tryptophan biosynthetic gene as a host, a nucleic acid fragment containing a loopout region and a tryptophan biosynthetic gene was introduced into the locus of the target gene. Thus, it was found that substitution between the target gene and the tryptophan biosynthetic gene can be confirmed, and that removal in the loopout of the tryptophan biosynthetic gene can be confirmed by growth in the presence of 5-FAA.

And surprisingly, the present inventor has established a double disruption in which tryptophan biosynthesis gene and pyrG gene are deleted as selection marker genes by setting the abundance of 5-FOA and 5-FAA to a predetermined concentration. We succeeded in producing and selecting transformed Aspergillus microorganisms. Aspergillus microorganisms such as Aspergillus soja and Aspergillus oryzae are known to have high resistance to drugs. Therefore, with respect to Aspergillus microorganisms, whether or not 5-FAA can be used for counter selection of tryptophan biosynthetic gene-deficient strains, and what level should be set for 5-FAA even if it can be used, etc. There has been no knowledge so far. In addition to this, it was assumed that the selection pressure was weak, that is, it was difficult to set a suitable drug concentration due to the small difference between sensitivity and tolerance. In spite of such technical background, the present inventor has produced and selected the above-mentioned double disruption transformed Aspergillus microorganism, and further by using the double disruption transformed Aspergillus microorganism, We succeeded in efficiently deleting two types of target nucleic acids on the chromosome in a short time. The present invention is an invention that has been completed based on such knowledge and successful examples.

Therefore, according to one aspect of the present invention, the following transformed Aspergillus microorganisms, compositions and methods of [1] to [8] are provided.
[1] Selection marker genes available to at least two markers Recycling Law on the chromosome deficient, a transformed Aspergillus (Aspergillus) microorganisms, the selectable marker gene, the tryptophan biosynthetic gene and tryptophan biosynthesis The transformed Aspergillus microorganism, comprising a gene different from the gene.
[2] A composition for transforming Aspergillus microorganisms, comprising at least two kinds of nucleic acid fragments comprising a loopout region and a selectable marker gene that can be used in a marker recycling method between homologous recombination regions, The said composition contains the nucleic acid fragment whose said selection marker gene is a tryptophan biosynthesis gene, and the nucleic acid fragment whose said selection marker gene is a gene different from a tryptophan biosynthesis gene.
[3] The transformed Aspergillus microorganism according to any one of [1] to [2], wherein the transformed Aspergillus microorganism is a Aspergillus microorganism other than Aspergillus aculeatus. Composition.
[4] The gene different from the tryptophan biosynthetic gene is a gene that complements the requirement of a nutrient substance and is involved in the biosynthesis of a toxic substance from an analogue of the nutrient substance [1] to [3] The transformed Aspergillus microorganism or composition according to any one of [3].
[5] The gene different from the tryptophan biosynthesis gene is any one of [1] to [4], which is a selectable marker gene selected from the group consisting of a uracil biosynthesis gene, a sulfate metabolism gene, and a nitrate metabolism gene. The transformed Aspergillus microorganism or composition according to Item.
[6] The tryptophan biosynthesis gene is a trpC gene, and the gene different from the tryptophan biosynthesis gene is at least one gene selected from the group consisting of a pyrG gene, a niaD gene, and an sC gene. ] The transformed Aspergillus microorganism or composition according to any one of [5] to [5].
[7] The first selection marker gene that can be used for the marker recycling method on the chromosome and the second selection marker gene that can be used for the marker recycling method are deficient, including the following steps (1) to (3) A method for producing a transformed Aspergillus microorganism, wherein one of the first selectable marker gene and the second selectable marker gene is a tryptophan biosynthetic gene and the other complements the requirement for a nutrient substance And a gene involved in biosynthesis of a toxic substance from the analog of the nutritional substance:
(1) Using a nucleic acid fragment containing a loop-out region and a first selectable marker gene between a homologous recombination region and a transformed Aspergillus microorganism lacking the first selectable marker gene on the chromosome, Obtaining a transformed Aspergillus microorganism by homologous recombination using the second selectable marker gene as a target;
(2) By culturing the transformed Aspergillus microorganism obtained in the step (1) in the presence of a nutrient substance corresponding to the second selection marker gene, the first selection marker on a chromosome Selecting a transformed Aspergillus microorganism in which the gene is inserted and the second selectable marker gene on the chromosome is deleted; and
(3) The transformed Aspergillus microorganism selected in the step (2), the nutrient substance corresponding to the first selectable marker gene, the analog of the nutrient substance, and the nutrient corresponding to the second selectable marker gene A step of selecting a transformed Aspergillus microorganism lacking the first selection marker gene and the second selection marker gene on a chromosome by culturing in the presence of a sex substance.
[8] Transformation using the first selection marker gene usable for the marker recycling method and the second selection marker gene usable for the marker recycling method, including the following steps (A) to (B) A method for deleting two types of target genes on the chromosome of an Aspergillus microorganism, wherein one of the first selectable marker gene and the second selectable marker gene is a tryptophan biosynthetic gene and the other is a nutrient The method, which is a gene that complements the requirement of a sexual substance and is involved in the biosynthesis of a toxic substance from an analogue of the nutrient substance:
(A) A transformed Aspergillus microorganism lacking the first selectable marker gene and the second selectable marker gene on the chromosome, the loop-out region and the first selection between the homologous recombination regions for the first target gene A first nucleic acid fragment comprising a marker gene and a second nucleic acid fragment comprising a loop-out region and a second selectable marker gene between a first nucleic acid fragment comprising a marker gene and a homologous recombination region for a second target gene; And obtaining a transformed Aspergillus microorganism by homologous recombination using the second target gene as a target; and (B) transforming Aspergillus microorganism obtained in the step (A) with the first Staining by culturing in the absence of a nutrient substance corresponding to the selectable marker gene and a nutrient substance corresponding to the second selectable marker gene The first selection marker gene and said second selection marker gene is a step of selecting transformed Aspergillus microorganism inserted above.
[9] The method according to [8], further comprising the following step (C):
(C) The transformed Aspergillus microorganism selected in the step (B) corresponds to the nutrient substance corresponding to the first selectable marker gene, the analog of the nutrient substance, and the second selectable marker gene. By culturing in the presence of a nutrient substance and an analogue of the nutrient substance, the first selectable marker gene, the second selectable marker gene, the first target gene, and the second target on a chromosome A step of selecting a transformed Aspergillus microorganism lacking the gene.
[10] The first selectable marker gene is a tryptophan biosynthetic gene, the nutrient substance analog corresponding to the first selectable marker gene is 5-FAA, and the concentration of 5-FAA is 0. 005% (w / v) to 0.02% (w / v), and
The second selectable marker gene is a pyrG gene, the analog of the nutrient substance corresponding to the first selectable marker gene is 5-FOA, and the concentration of the 5-FOA is 0.05% (w / The method according to [9], wherein v) to 0.15% (w / v).
[11] The method according to any one of [7] to [10], wherein the transformed Aspergillus microorganism is a Aspergillus microorganism other than Aspergillus acreatas.

According to the transformed Aspergillus genus microorganism and method which are one aspect of the present invention, at least two kinds of target nucleic acids on the chromosome of the transformed Aspergillus bacterium are efficiently and quickly deleted using the marker recycling method. be able to. According to the composition and method of one embodiment of the present invention, a transformed Aspergillus genus microorganism lacking at least two selectable marker genes that can be used in the marker recycling method can be produced. By applying the transformed Aspergillus microorganism, composition and method which is one embodiment of the present invention, it can be expected to rapidly detect a new phenotype by simultaneous disruption of genes having similar or related structures or functions.

FIG. 1 is a diagram showing an overview of the tryptophan biosynthesis pathway of yeast. FIG. 2 is a diagram showing an outline of transformation of an Aspergillus soja KP-del strain using an AstrpC disruption cassette as described in Examples described later. FIG. 3 is a diagram showing an outline of transformation of an AstrpC-disrupted strain using an Asparp1 disruption cassette as described in Examples described later. FIG. 4A is a diagram showing the results of agarose gel electrophoresis for selecting Asparp1-disrupted strains, as described in the Examples described later. FIG. 4B is a diagram showing the results of agarose gel electrophoresis for selecting Asparp1-disrupted strains as described in the examples described later. FIG. 5 shows the outline of the selection procedure of a strain from which the trpC gene and parp1 gene have been removed, and the 5-FAA resistance, as described in the examples described later, in which the trpC gene introduced into the Asparp1-disrupted strain was removed by loop-out. It is a figure which shows the appearance frequency of a strain | stump | stock. FIG. 6A is a diagram showing the results of agarose gel electrophoresis for selection of AstrC-removed strains as described in Examples described later. FIG. 6B is a diagram showing the results of agarose gel electrophoresis for selection of AstrC-removed strains as described in Examples described later. FIG. 7 is a diagram showing an outline of transformation of an AotrpC disrupted strain using an AohypG disruption cassette as described in the examples described later. FIG. 8 is a diagram showing an outline of a selection procedure of a strain from which the trpC gene and the hypG gene have been removed, in which the trpC gene introduced into the AohypG disrupted strain is removed by loop-out, as described in the examples described later. is there. FIG. 9A is a diagram showing the results of agarose gel electrophoresis for selection of AotrpC-removed strains as described in Examples described later. FIG. 9B is a diagram showing the results of agarose gel electrophoresis for selection of AotrpC-removed strains as described in Examples described later. FIG. 10 is a diagram showing an outline of a procedure for producing an AspyrG. AsrpC double disruption strain as described in Examples described later. FIG. 11 is a diagram showing an outline of the procedure from the production of an Asparp1 · Asnph double disruption strain to the production of an AspyrG · AstropC double removal strain as described in the Examples described later. FIG. 12 is a diagram showing the results of agarose gel electrophoresis for selection of Asparp1 / Asnph double disruption strains as described in the Examples described later. FIG. 13A is a diagram showing the results of agarose gel electrophoresis for selection of AspyrG / AstrpC double-removed strains as described in Examples described later. FIG. 13B is a diagram showing the results of agarose gel electrophoresis for selection of AspyrG and AsrpC double-removed strains as described in the examples described later. FIG. 13C is a diagram showing the results of agarose gel electrophoresis for selection of AspyrG and AsrpC double-removed strains as described in Examples described later.

Hereinafter, although the details of the transformed Aspergillus microorganisms, compositions and methods which are one embodiment of the present invention will be described, the technical scope of the present invention is not limited only to the matters of this item, the present invention As long as the purpose is achieved, various modes can be taken.

Each term in this specification is used in the meaning normally used by those skilled in the art unless otherwise specified, and should not be construed as having an unduly limiting meaning.

For example, “and / or” means any one of a plurality of related items listed, or any combination or all combinations of two or more.

“Gene deficiency” means that a gene is normal, such as the gene is not normally transcribed due to the loss of part or all of the gene, or the transcribed protein does not perform its original protein function. It means that gene expression is hindered without functioning. In the present specification, gene deletion and gene disruption are used synonymously.

“Expression of a gene” means that a protein encoded by a gene is produced in a form having an original structure or activity through transcription or translation.

A “selection marker gene” is a gene that causes a trait to be used as a means for selecting transformants, so that transformants can be specifically selected in the presence or absence of a corresponding selective substance. What makes it possible to prevent or not. “Function of a selectable marker gene” means that a transformant can be specifically selected in the presence or absence of a corresponding selective substance. For example, when the selectable marker gene is a drug resistance gene, the transformant is specifically selected in the presence of the drug by the function of the selectable marker gene. Further, when the selectable marker gene is an auxotrophic gene, the function of the selectable marker gene is exerted so that the transformant is specifically selected in the absence of the nutrient substance. Among selectable marker genes, “selectable marker genes that can be used in the marker recycling method” can be used for counter selection that makes it impossible to grow in an environment containing a specific drug when the gene is expressed. A selectable marker gene.

“Homologous recombination region” refers to nucleic acids that are homologous to regions on both sides of the target gene (target gene) on the chromosome (that is, upstream (5 ′ end side) and downstream (3 ′ end side)). An array. Of the “homologous recombination region”, a region upstream of the target gene is called “homologous recombination upstream region”, and a region downstream of the target gene is called “homologous recombination downstream region”.

“Loop out” refers to a phenomenon in which homologous nucleic acid sequences on the same chromosome undergo recombination and the nucleic acid sequences between them are lost. The “loop-out region” is a region that enables loop-out. For example, it refers to a region for removing a selectable marker gene introduced together with the loop-out region by loop-out.

“Biosynthetic gene” means one or more genes that express a protein that functions in the biosynthetic pathway of a target substance. For example, an enzyme that catalyzes a reaction for conversion to a target substance. Examples include genes that are expressed.

“Metabolic gene” means one or more genes that express a protein that functions in the metabolic pathway of a target substance. For example, it catalyzes a reaction that converts a target substance into another substance. Examples include genes that express enzymes.

(Overview of transformed Aspergillus microorganisms and compositions)
Transformation Aspergillus (Aspergillus) microorganism which is one embodiment of the present invention, as two or selectable marker genes available more marker Recycling Law deficient in the chromosomal Aspergillus microorganism is a host organism A microorganism of the genus Aspergillus transformed with a host organism. The composition which is one embodiment of the present invention is used to destroy a target gene in the transformed Aspergillus microorganism which is one embodiment of the present invention, and a loop-out region and a marker recycling method between homologous recombination regions. At least two or more types of nucleic acid fragments containing a selectable marker gene.

In the transformed Aspergillus microorganism and composition of one embodiment of the present invention, one or more selectable marker genes that can be used in the marker recycling method are tryptophan biosynthesis genes, and the selectable marker that can be used in the marker recycling method Another one type or two or more types of genes are genes different from tryptophan biosynthesis genes.

(Selectable marker genes that can be used in the marker recycling law)
An overview of the yeast tryptophan biosynthetic pathway is shown in FIG. As shown in FIG. 1, in the biosynthesis of tryptophan, TRP4 (anthropilate phosphoryltransferase), TRP1 (phosphorylanthranylate isomerase), TRP3 (indole-3-glycerol phosphatase) Is involved. The reaction from chorismate to anthranilate is catalyzed by TRP2 (Anthranilate synthase).

On the other hand, the present inventor has found that a gene encoding an enzyme corresponding to TRP4 is in the region of 1554760-1553403 of scaffold00063 from the public genome database (BioProject Accession: PRJDA60265) of Aspergillus sojae ( A. sojae ) NBRC4239 strain. Predicted that there was. When an attempt was made to destroy the region, only a strain in which a chromosome that destroyed the region and a non-destructed chromosome were mixed was obtained, and as a result, a strain that destroyed the region was not obtained.

The gene encoding the enzyme corresponding to TRP5 was predicted to be in the region of 1470936-1473260 of scaffold0660, the region of 917995-920152 of scaffold056, the region of 350582-352846 of scaffold057, and the region of 662605-659898 of scaffold00001. Thus, it was predicted that there were four genes encoding the enzyme corresponding to TRP5, and it was abandoned because it was very difficult to destroy all of them.

Furthermore, in Aspergillus soja NBRC4239 strain, genes encoding enzymes corresponding to the respective enzymes of TRP1 and TRP3 were not found. On the other hand, a gene encoding an enzyme having the functions of TRP1 and TRP3 was predicted to be in the region 1213700-1211376 of scaffold0408, and this region was named AstrpC gene. And in Aspergillus soja, it succeeded in obtaining the transformed microorganism of the genus Aspergillus which cannot biosynthesize tryptophan from anthranilic acid by destroying the AstrpC gene.

Based on the above circumstances, the tryptophan biosynthetic gene as a selectable marker gene that can be used in the marker recycling method is preferably a gene encoding an enzyme having the functions of TRP1 and TRP3 of yeast, ie, the trpC gene. It may be a gene encoding an enzyme corresponding to TRP4, TRP5 or TRP2 of yeast. A tryptophan biosynthetic gene may be used alone or in combination of two or more of the above genes.

The gene different from the tryptophan biosynthetic gene is not particularly limited as long as it is a selectable marker gene that can be used in a marker recycling method different from the tryptophan biosynthetic gene, and examples thereof include a drug resistance gene and an auxotrophic gene. The auxotrophic gene is not particularly limited as long as it is a gene that complements the auxotrophy of the host organism, and examples thereof include a pyrG gene, a niaD gene, and an sC gene. As the gene different from the tryptophan biosynthesis gene, one of the above genes can be used alone, or two or more can be used in combination. The strain lacking the niaD gene is resistant to chlorate, and the strain lacking the sC gene is resistant to selenate.

The pyrG gene, the trpC gene, the niaD gene, and the sC gene are registered in the NCBI GeneBank for each derived organism. For example, Table 1 shows GeneBank accession numbers of the pyrG gene, the trpC gene, the niaD gene and the sC gene of Aspergillus soja, Aspergillus oryzae and Aspergillus niger. For Aspergillus soja, the scaffold on the chromosome is shown. A person skilled in the art can obtain the nucleotide sequences of these genes of Aspergillus microorganisms by referring to NCBI GeneBank.

The gene different from the tryptophan biosynthetic gene is preferably an auxotrophic gene from the viewpoint of ease of selection of transformants among the above-mentioned genes. An auxotrophic gene includes a biosynthetic gene and a metabolic gene. Among the auxotrophic genes, it is more preferable that the gene complements the requirement of the nutrient substance and is involved in the biosynthesis of the toxic substance from the analogue of the nutrient substance. The uracil biosynthesis gene, sulfate More preferred are metabolic genes and nitrate metabolic genes, and even more preferred are pyrG gene, niaD gene and sC gene.

The selectable marker gene that can be used in the marker recycling method may not be completely the same as the gene originally possessed by the derived organism having the selectable marker gene that can be used in the marker recycling method (ie, the wild type gene), and at least the wild type As long as the gene expresses a protein having the same or similar enzymatic properties as the protein expressed by the gene (ie, wild-type protein), the base sequence complementary to the base sequence of the wild-type gene and a stringent sequence are used. It may be DNA having a base sequence that hybridizes under conditions.

The term “base sequence that hybridizes under stringent conditions” in the present specification refers to a colony hybridization method, plaque hybridization method, Southern blot hybridization method using a DNA having a base sequence of a wild-type gene as a probe. It means the base sequence of DNA obtained by using.

The term “stringent conditions” in the present specification is a condition in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal. The hybridization system used, the type of probe, and the sequence It depends on the length. Such conditions can be determined by changing the hybridization temperature, washing temperature and salt concentration. For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the hybridization and washing temperature and, if necessary, lowering the washing salt concentration. If no specific hybrid signal is detected, the hybrid can be stabilized by lowering the hybridization and washing temperatures and, if necessary, raising the washing salt concentration.

As a specific example of stringent conditions, for example, a DNA probe is used as a probe, and hybridization is 5 × SSC, 1.0% (w / v). Nucleic acid hybridization blocking reagent (Roche Diagnostics) 0.1% (w / v) N-lauroyl sarcosine, 0.02% (w / v) SDS is used overnight (about 8 to 16 hours). Washing is performed using 0.1-0.5 × SSC, 0.1% (w / v) SDS, preferably 0.1 × SSC, 0.1% (w / v) SDS, twice for 15 minutes. Do. The temperature for performing hybridization and washing is 65 ° C or higher, preferably 68 ° C or higher.

In addition, as the DNA having a base sequence that hybridizes under stringent conditions, for example, using a DNA having a base sequence of a wild-type gene derived from a colony or plaque or a filter on which the DNA fragment is immobilized, After performing hybridization at 40 to 75 ° C. in the presence of DNA obtained by hybridization under the stringent conditions and 0.5 to 2.0 M NaCl, preferably 0.7 to 1. After hybridization at 65 ° C. in the presence of 0 M NaCl, 0.1 to 1 × SSC solution (1 × SSC solution is 150 mM sodium chloride, 15 mM sodium citrate) at 65 ° C. Examples thereof include DNA that can be identified by washing the filter. Probe preparation and hybridization methods are described in Molecular Cloning: A laboratory Manual, 2nd-Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 (hereinafter these documents may be referred to as reference technical documents). Can do. In addition to the conditions such as the salt concentration and temperature of the buffer, those skilled in the art will consider other conditions such as probe concentration, probe length, reaction time, etc. Conditions for obtaining a DNA having a base sequence that hybridizes under stringent conditions with a complementary base sequence can be appropriately set.

Examples of DNA containing a base sequence that hybridizes under stringent conditions include DNA having a certain sequence identity with a base sequence of a DNA having a base sequence of a wild-type gene used as a probe. 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more , 98% or more or 99% or more, more preferably 99.5% or more of DNA having sequence identity. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

The base sequence that hybridizes with the base sequence complementary to the base sequence of the wild-type gene under stringent conditions is, for example, as follows: 1 to several, preferably 1 to 20, more preferably 1 to 15, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 per unit. Includes base sequences with base deletions, substitutions, additions, and the like. Here, “base deletion” means that there is a deletion or disappearance in the base in the sequence, and “base replacement” means that the base in the sequence is replaced with another base, “Addition of a base” means that a new base is added to be inserted.

The protein encoded by the base sequence that hybridizes with the base sequence complementary to the base sequence of the wild-type gene under stringent conditions is 1 to several in the amino acid sequence of the protein encoded by the base sequence of the wild-type gene. Although it is likely to be a protein having an amino acid sequence having deletion, substitution, addition, etc. of individual amino acids, it has the same enzyme activity as the protein encoded by the base sequence of the wild-type gene.

A protein having the same or similar enzymatic properties as the wild-type protein has an amino acid sequence having a deletion, substitution, addition, etc. of one to several amino acids in the amino acid sequence of the wild-type protein It may consist of. Here, the range of “1 to several” in “deletion, substitution and addition of 1 to several amino acids” of the amino acid sequence is not particularly limited, but for example, the unit of 100 amino acids in the amino acid sequence is one unit. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 per unit, preferably Means about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably about 1, 2, 3, 4 or 5. In addition, “amino acid deletion” means deletion or disappearance of an amino acid residue in the sequence, and “amino acid substitution” means that an amino acid residue in the sequence is replaced with another amino acid residue. “Addition of amino acid” means that a new amino acid residue is added to the sequence.

A specific embodiment of “deletion, substitution, addition of 1 to several amino acids” includes an embodiment in which one to several amino acids are replaced with another chemically similar amino acid. For example, a case where a certain hydrophobic amino acid is substituted with another hydrophobic amino acid, a case where a certain polar amino acid is substituted with another polar amino acid having the same charge, and the like can be mentioned. Such chemically similar amino acids are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of the basic amino acid having a positive charge include arginine, histidine, and lysine. Examples of acidic amino acids having a negative charge include aspartic acid and glutamic acid.

Examples of amino acid sequences having a deletion, substitution, addition, etc. of one to several amino acids in the amino acid sequence of the wild type protein include amino acid sequences having a certain sequence identity with the amino acid sequence of the wild type protein. For example, 80% or more, preferably 85% or more, more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% with the amino acid sequence of the wild-type protein As mentioned above, amino acid sequences having sequence identity of 97% or more, 98% or more, or 99% or more, and more preferably 99.5% or more can be mentioned. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

(Means for calculating sequence identity)
The method for determining the sequence identity of the base sequence or amino acid sequence is not particularly limited. For example, by using a commonly known method, the amino acid sequence of the wild type gene or the wild type protein expressed by the wild type gene and the target base It is obtained by aligning sequences and amino acid sequences and using a program for calculating the coincidence ratio between the sequences.

As a program for calculating the coincidence rate in two base sequences or amino acid sequences, for example, the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993), and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST, which is a program for determining sequence identity with higher sensitivity than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Therefore, a person skilled in the art can search, for example, a sequence showing high sequence identity from a database using a program described above. These are available, for example, at the National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Each of the above methods can be generally used to search a sequence showing sequence identity from a database. As a means for determining the sequence identity of individual sequences, Genetyx network version version 12.0. 1 (Genetics) homology analysis can also be used. This method is based on the Lipman-Pearson method (Science 227: 1435-1441, 1985). When analyzing the sequence identity of a base sequence, a region encoding a protein (CDS or ORF) is used if possible.

(Origin of selectable marker genes that can be used in the marker recycling law)
The selectable marker gene that can be used in the marker recycling method is derived from, for example, a biological species that can confirm the expression of a selectable marker gene that can be used in the marker recycling method or the function as a selectable marker gene that can be used in the marker recycling method. Examples of the organism derived from the selectable marker gene that can be used in the marker recycling method include microorganisms and the like, and preferably Aspergillus microorganisms that are the same as or closely related to the host organism. Specific examples of the genus Aspergillus microorganisms, Aspergillus sojae (Aspergillus sojae), Aspergillus oryzae (Aspergillus oryzae), Aspergillus niger (Aspergillus niger), Aspergillus tamari (Aspergillus tamarii), Aspergillus Ruchuenshisu (Aspergillus luchuensis), Aspergillus Usami (Aspergillus usamii), Aspergillus Akureatasu (Aspergillus aculeatus), Aspergillus saitoi (Aspergillus saitoi), Aspergillus nidulans (Aspergillus nidulans) is like .

Among the specific examples of the microorganisms belonging to the genus Aspergillus, Aspergillus soya, Aspergillus oryzae, Aspergillus niger, Aspergillus tamari, Aspergillus luchuensis, Aspergillus usami, Aspergillus acreatus and Aspergillus cytoy are miso, soy sauce, It has an extensive track record in the production of sake, shochu and other brewed foods, citric acid, and amylase and other enzyme preparations, and is used industrially due to its high enzyme productivity and high reliability for safety over many years. It is a possible microorganism.

A selectable marker gene that can be used in the marker recycling method is introduced into a host organism and transformed to express a selectable marker gene protein that can be used in the marker recycling method. Will come to present. Therefore, a selectable marker gene protein that can be used in the marker recycling method expressed in the transformant can be used in the marker recycling method in order to exhibit the selection marker function without being inactivated by the growth conditions of the host organism. The organism from which the selectable marker gene is derived is preferably an Aspergillus microorganism that has similar growth conditions to the host organism to be transformed.

(Host organism)
The host organism is particularly limited as long as it is a microorganism belonging to the genus Aspergillus that has a selectable marker gene that can be used for the marker recycling method on the chromosome and that can function by the selectable marker gene by deleting the selectable marker gene. Not. However, the microorganism of the genus Aspergillus is preferably an microorganism of the genus Aspergillus other than Aspergillus acreatus. -More preferred are Aspergillus microorganisms such as Luciensis, Aspergillus usami, Aspergillus cytoii, Aspergillus nidulans.

The host organism may be a wild strain or a transformant obtained by transforming a wild strain in advance. The transformant previously transformed with a wild strain used as a host organism is not particularly limited.

For example, since Aspergillus microorganisms tend to have a low homologous recombination frequency, when a transformant is produced by homologous recombination, a ku encoding a protein such as Ku70 or Ku80 involved in the heterologous recombination mechanism. It is preferable to use transformed Aspergillus microorganisms whose genes are suppressed.

Such suppression of the ku gene can be performed by any method known to those skilled in the art. For example, the ku gene is disrupted using a ku gene disruption vector, or an antisense expression vector for the ku gene. This can be achieved, for example, by inactivating the ku gene by an anti-sense RNA method using the It is also possible to destroy the ku gene by a genome editing technique using Cas nuclease and a guide RNA targeting the ku gene. The transformed Aspergillus microorganism thus obtained has a significantly increased homologous recombination frequency compared to the original Aspergillus microorganism before genetic manipulation relating to suppression of the ku gene. Specifically, it is elevated at least 2 times, preferably at least 5 times, preferably at least 10 times, preferably at least about 50 times.

(One embodiment of transformant)
One embodiment of the transformant is that the host organism is Aspergillus soja, Aspergillus oryzae, Aspergillus niger, Aspergillus tamari, Aspergillus luchuensis, Aspergillus usami or Aspergillus cytoii and trpC gene on the chromosome Although it is a transformed Aspergillus microorganism lacking a gene, it is not limited thereto.

(Method for producing transformed Aspergillus microorganism)
A method for producing a transformed Aspergillus microorganism lacking a selectable marker gene that can be used in the marker recycling method is not particularly limited. For example, a selectable marker gene that can be used in the marker recycling method on the chromosome of the host organism according to a conventional method And a method including a step of deleting a selectable marker gene that can be used in the marker recycling method by inserting or replacing a foreign nucleic acid fragment at the locus of the selectable marker gene. This step is preferably performed using homologous recombination.

In the method using homologous recombination, for example, a foreign nucleic acid fragment is constructed to be linked between the homologous recombination region homologous to the upstream region and the downstream region of the selectable marker gene that can be used for the marker recycling method on the chromosome. For example, a method of introducing a selected nucleic acid fragment into a host organism and substituting a selection marker gene that can be used for a marker recycling method on a chromosome by homologous recombination with a foreign nucleic acid fragment, and the like. In the present specification, a nucleic acid fragment prepared for transforming a host organism may be referred to as a transformation cassette. The foreign nucleic acid fragment is preferably a selectable marker gene that can be used in the marker recycling method different from the selectable marker gene that can be used in the marker recycling method to be deleted.

The transformation cassette preferably contains a loop-out region. In a transformation cassette containing a loop-out region and a foreign nucleic acid fragment, homology is obtained if the region having the same sequence as the loop-out region is upstream or downstream of the site capable of being introduced by homologous recombination on the chromosome of the host organism. After the transformation cassette is introduced onto the chromosome of the host organism by recombination, homologous recombination occurs between the loopout region upstream or downstream of the loopout region and a homologous region, thereby introducing the introduced foreign Nucleic acid fragments can be removed by loop out. By taking such means, a transformant from which the foreign nucleic acid fragment has been removed after introducing the foreign nucleic acid fragment into the gene locus of the selectable marker gene can be obtained.

One embodiment of the cassette for transformation is, for example, a nucleic acid fragment containing a loop-out region and a foreign nucleic acid fragment between homologous recombination regions. The nucleic acid fragment is obtained by polymerase chain reaction (hereinafter referred to as “PCR”) of DNA fragments in the upstream region of the homologous recombination, the loop-out region and the downstream region of the homologous recombination according to a conventional method using the chromosomal DNA of Aspergillus microorganisms as a template. For each of the constructs obtained by sequentially connecting the homologous recombination upstream region, the loopout region, the foreign nucleic acid (DNA) fragment, and the homologous recombination downstream region to the In-Fusion Cloning Site at the multicloning site of the plasmid pUC19. It can be obtained by preparing a plasmid and then amplifying it by PCR using the resulting plasmid for construction as a template DNA.

The method for extracting chromosomal DNA is not particularly limited. For example, culturing Aspergillus microorganisms, removing moisture from the obtained cells, and physically grinding them using a mortar while cooling in liquid nitrogen Thus, a fine powdery cell piece is obtained, and a chromosomal DNA fraction is extracted from the cell piece by a usual method. A commercially available chromosomal DNA extraction kit such as DNeasy Plant Mini Kit (Qiagen) can be used for the chromosomal DNA extraction operation.

As a method for transforming Aspergillus microorganisms, a method known to those skilled in the art can be appropriately selected. For example, after preparing a protoplast of a host organism, a protoplast PEG method using polyethylene glycol and calcium chloride (for example, Mol. Gen. Genet. 218, 99-104, 1989, Japanese Patent Application Laid-Open No. 2007-2222055, etc.) can be used. As a medium for regenerating Aspergillus microorganisms, an appropriate medium is used according to the host organism to be used, the selection marker gene to be deleted, and the introduced foreign nucleic acid fragment. For example, when Aspergillus soya is used as the host organism and a drug resistance gene is used as the foreign nucleic acid fragment, the transformant can be regenerated using, for example, a minimal agar medium containing the corresponding drug.

Confirmation that a transformed Aspergillus microorganism lacking the selectable marker gene that can be used in the marker recycling method was produced was obtained under the condition that the function of the selectable marker gene that can be used in the defective marker recycling method was confirmed. Can be performed by culturing and confirming the presence or absence of growth. For example, when the selection marker genes that can be used in the deficient marker recycling method are trpC gene and pyrG gene, in the presence of 5-fluoroanthranilic acid (5-FAA) and 5-fluoroorotic acid (5-FOA) By confirming the growth of, it can be confirmed that a transformed Aspergillus microorganism is produced.

In addition, confirmation that the transformed Aspergillus spp. Microorganism was produced is that a chromosomal DNA is extracted from the transformant, PCR is performed using this as a template, and a PCR product that can be amplified when transformation occurs occurs. You may carry out by confirming.

For example, a forward primer complementary to a region located further upstream from the upstream region of homologous recombination incorporated in the transformation cassette and a region located further downstream from the downstream region of homologous recombination incorporated into the transformation cassette It is preferable to perform PCR in combination with a typical reverse primer and confirm that a product of the expected length is produced when homologous recombination occurs.

By transforming a transformant lacking the first selectable marker gene that can be used in the marker recycling method, the second selectable marker gene that can be used in the marker recycling method is further deleted, so that two types of marker recycling methods can be obtained. A transformant lacking an available selectable marker gene can be prepared. By repeating this procedure, a transformant lacking a selectable marker gene that can be used in a plurality of marker recycling methods can be produced.

For example, when the selectable marker genes available for the marker recycling method are trpC gene and pyrG gene, a loop-out region and a pyrG gene between homologous recombination regions using a transformed Aspergillus microorganism lacking the pyrG gene as a host organism. Is transformed so as to lack the trpC gene, and the introduced pyrG gene is removed by loop-out, thereby obtaining a transformed Aspergillus microorganism lacking the trpC gene and the pyrG gene. Examples of transformed Aspergillus microorganisms lacking the pyrG gene include the Aspergillus soja KP-del strain described in Example 2 of Japanese Patent No. 626139.

Specific examples of the method for producing transformed Aspergillus microorganisms include, but are not limited to, the method described in Example 2 of Japanese Patent No. 6261039. According to this method, a strain in which the auxotrophic gene is inactivated by natural mutation is obtained from a wild strain of an Aspergillus microorganism. The auxotrophic gene may or may not be a selectable marker gene that can be used in the marker recycling method to be deleted.

If the auxotrophic gene is a selectable marker gene that can be used in the marker recycling method to be deleted, the tryptophan biosynthesis gene is deleted by introducing the auxotrophy gene into the tryptophan biosynthesis gene locus, Subsequently, by removing the auxotrophic gene by loop-out, a transformed Aspergillus genus microorganism lacking a selectable marker gene that can be used for two types of marker recycling methods on the chromosome is obtained.

If the auxotrophic gene is not a selectable marker gene that can be used for the marker recycling method to be deleted, for example, the gene of the selectable marker gene that can be used for the marker recycling method that is different from the tryptophan biosynthesis gene. By introducing into the locus, a selectable marker gene that can be used for a marker recycling method that is different from the tryptophan biosynthesis gene is deleted, and then a selectable marker gene that can be used for a marker recycling method that is different from the tryptophan biosynthesis gene is tryptophan biosynthesis By introducing the gene locus, the tryptophan biosynthesis gene is deleted, and then the selectable marker gene that can be used for the marker recycling method different from the introduced tryptophan biosynthesis gene is removed by loop-out. Ri, two selectable marker genes available marker Recycling Law on the chromosome deficient, can be obtained transformant Aspergillus microorganism. For example, by introducing the above auxotrophic gene into the tryptophan biosynthetic gene locus, the tryptophan biosynthetic gene can be deleted, and then the tryptophan biosynthetic gene can be used in a marker recycling method different from the tryptophan biosynthetic gene. By introducing the gene into a selectable marker gene locus, the selectable marker gene that can be used for the marker recycling method different from the tryptophan biosynthetic gene is deleted, and then the introduced tryptophan biosynthetic gene is removed by loop-out, A transformed Aspergillus microorganism can be obtained that lacks a selectable marker gene that can be used for two types of marker recycling methods on the chromosome.

Another aspect of the present invention is a method for producing a transformed Aspergillus microorganism which is an aspect of the present invention. The production method of one embodiment of the present invention includes at least the following steps. However, in the following process, one of the first selectable marker gene and the second selectable marker gene that can be used in the marker recycling method is a tryptophan biosynthetic gene, and the other complements the requirement for a nutrient substance. And a gene involved in biosynthesis of a toxic substance from the analog of the nutrient substance.
(1) A nucleic acid fragment comprising a transformed Aspergillus microorganism lacking a first selectable marker gene that can be used for the marker recycling method on a chromosome, comprising a loopout region and a first selectable marker gene between homologous recombination regions (2) Obtaining transformed Aspergillus microorganisms by homologous recombination using a second selectable marker gene that can be used in the marker recycling method on chromosome as a target (2) obtained in the above step (1) By culturing the transformed Aspergillus microorganism in the presence of a nutritional substance corresponding to the second selectable marker gene, the first selectable marker gene is inserted on the chromosome, and the second selectable on the chromosome. Step (3) for selecting transformed Aspergillus microorganisms lacking the selection marker gene of (3) selected in the above step (2) By culturing the transformed Aspergillus microorganism in the presence of a nutrient substance corresponding to the first selectable marker gene and an analog of the nutrient substance and a nutrient substance corresponding to the second selectable marker gene, Selecting a transformed Aspergillus microorganism lacking the first selection marker gene and the second selection marker gene

(Target gene deletion method)
When the transformed Aspergillus microorganism of one embodiment of the present invention is used, two or more types of target genes on the chromosome of the transformed Aspergillus microorganism can be deleted simultaneously or stepwise.

Another embodiment of the present invention is a method for deleting two types of target genes on the chromosome of the transformed Aspergillus microorganism of one embodiment of the present invention. The target gene deletion method of one embodiment of the present invention includes at least the following steps (A) to (B). However, in the following process, one of the first selectable marker gene and the second selectable marker gene that can be used in the marker recycling method is a tryptophan biosynthetic gene, and the other complements the requirement for a nutrient substance. And a gene involved in biosynthesis of a toxic substance from the analog of the nutrient substance.
(A) Homologous to a first target gene, a transformed Aspergillus microorganism lacking the first selectable marker gene that can be used for the marker recycling method on the chromosome and the second selectable marker gene that can be used for the marker recycling method A first nucleic acid fragment comprising a loopout region and a first selectable marker gene between recombination regions and a first nucleic acid fragment comprising a loopout region and a second selectable marker gene between homologous recombination regions for a second target gene; Using the nucleic acid fragment of 2 to obtain a transformed Aspergillus microorganism by homologous recombination using the first target gene and the second target gene as targets; and
(B) In the absence of the nutrient substance corresponding to the first selectable marker gene and the nutrient substance corresponding to the second selectable marker gene, the transformed Aspergillus genus microorganism obtained in the step (A) A step of selecting a transformed Aspergillus microorganism in which the first selection marker gene and the second selection marker gene are inserted on a chromosome by culturing in step (1).

The target gene deletion method of one embodiment of the present invention preferably further includes the following step (C):
(C) The transformed Aspergillus microorganism selected in the step (B) corresponds to the nutrient substance corresponding to the first selectable marker gene, the analog of the nutrient substance, and the second selectable marker gene By culturing in the presence of a nutrient substance and an analogue of the nutrient substance, the first selectable marker gene, the second selectable marker gene, the first target gene, and the second target on a chromosome A step of selecting a transformed Aspergillus microorganism lacking the gene.

If two or more target genes on the chromosome of a transformed Aspergillus microorganism are desired to be deleted stepwise, a first nucleic acid fragment for the first target gene, a first selectable marker that can be used in the marker recycling method The above steps (A) to (C) are performed using a nutritional substance corresponding to the gene, an analog of the nutritional substance, and a nutritional substance corresponding to the second selection marker gene that can be used in the marker recycling method. After that, the second nucleic acid fragment for the second target gene, the nutrient substance corresponding to the second selectable marker gene and the analog of the nutrient substance, and the nutrient substance corresponding to the first selectable marker gene are used. And what is necessary is just to implement the said process (A)-(C). Further, the step (C) can be performed stepwise by dividing it into a step of removing the first selectable marker gene and a step of removing the second selectable marker gene. Such a stepwise target gene deletion method is also included in the target gene deletion method of one embodiment of the present invention.

(Specific example of method)
In the non-limiting specific example of the production method of one embodiment of the present invention and the target gene deletion method of one embodiment of the present invention, the first selectable marker gene that can be used in the marker recycling method is a pyrG gene, The nutritional substance corresponding to the selection marker gene is uracil / uridine, the analog of the nutritional substance corresponding to the first selection marker gene is 5-FOA, and the second selection marker that can be used in the marker recycling method The gene is a trpC gene, the nutrient substance corresponding to the second selectable marker gene is tryptophan, and the analog of the nutrient substance corresponding to the second selectable marker gene is 5-FAA.

In another non-limiting example of the production method of one embodiment of the present invention and the target gene deletion method of one embodiment of the present invention, the first selectable marker gene that can be used in the marker recycling method is a trpC gene, The nutritional substance corresponding to the first selection marker gene is tryptophan, the analog of the nutritional substance corresponding to the first selection marker gene is 5-FAA, and the second selection marker that can be used in the marker recycling method The gene is a pyrG gene, the nutrient substance corresponding to the second selectable marker gene is uracil / uridine, and the analog of the nutrient substance corresponding to the second selectable marker gene is 5-FOA.

In the above specific examples, the concentrations of 5-FAA and 5-FOA used for selection of strains resistant to 5-FAA, strains resistant to 5-FOA, and strains resistant to 5-FAA and 5-FOA It is a finding for the first time by the present inventors that these are different. The concentrations of 5-FAA and 5-FOA used for selection of strains resistant to 5-FAA and 5-FOA are preferably lower than those used alone. For selection of a strain having resistance to 5-FAA, 0.03% (w / v) to 0.05% (w / v) 5-FAA is preferably used. For selection of a strain having resistance to 5-FOA, 0.2% (w / v) to 0.4% (w / v) 5-FOA is preferably used. For selection of strains resistant to 5-FAA and 5-FOA, 0.005% (w / v) to 0.02% (w / v) 5-FAA and 0.05% (w / v) to It is preferable to use 0.15% (w / v) 5-FOA.

The target gene in the target gene deletion method of one embodiment of the present invention is not particularly limited, and may be appropriately set depending on the purpose of research or investigation. For example, as described in the Examples described later, when the host organism is Aspergillus soya, a double disruption strain in which these genes are disrupted can be obtained using the parp1 gene and the nph gene as target genes.

In addition, when the target gene deletion method of one embodiment of the present invention is applied, a gene that can be used for two types of marker recycling methods cannot be achieved only by using a gene that can be used for one type of marker recycling method. It becomes possible by using it. For example, using a transformed Aspergillus microorganism lacking the pyrG gene and trpC gene, the trpC gene is introduced into the locus of the ku gene to produce a ku disrupted strain. At this time, not only the nucleic acid fragment containing the trpC gene can be used between the homologous recombination regions, but also two kinds of nucleic acids in which the fragments are divided so as to partially overlap each other in the middle of the trpC gene. Fragments prepared and mixed can be used for ku gene disruption. Next, after introducing and removing the pyrG gene as a target gene into the locus of the protease gene, if necessary, the introduction and removal of the pyrG gene from another protease locus is repeated, and then the introduced trpC gene is removed. Restore the ku gene. At this time, by partially overlapping the ku gene, it is possible to restore the ku gene together with the removal of the trpC gene by loop-out. A gene expression cassette that expresses the desired protein is introduced into the resulting protease gene-disrupted strain at random by non-homologous recombination, so that a strain that highly expresses the protein while preventing degradation by the protease can be obtained. Obtainable.

In the production method of one embodiment of the present invention and the method for deleting a target gene of one embodiment of the present invention, as long as the problems of the present invention can be solved, various steps and operations are performed before, after, or during the above-described steps. You can join.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples, and the present invention can take various modes as long as the problems of the present invention can be solved. .

The primers used in this example are shown in Tables 2A to 2B. Of the sequences in the table, lower case sequences indicate additional sequences for ligation to adjacent fragments.

[Example 1. Use of the trpC gene as a selectable marker gene that can be used in the marker recycling method in Aspergillus sojae]
1. Preparation of AsstrC disruption strain (1-1) Extraction of chromosomal DNA Polypeptone dextrin medium (1% (w / v) polypeptone, 2% (w / v) dextrin, 0.5% (w / v) in a 150 ml Erlenmeyer flask ) KH ₂ PO ₄ , 0.1% (w / v) NaNO ₃ , 0.05% (w / v) MgSO ₄ .7H ₂ O, 0.1% (w / v) casamino acid; pH 6.0) Was prepared with distilled water, inoculated with conidia of Aspergillus soja NBRC4239 strain, and cultured with shaking at 30 ° C. overnight.

The bacterial cells were collected from the obtained culture broth by filtration, and the water was removed by sandwiching between paper towels. The bacterial cells were pulverized while being cooled with liquid nitrogen using a mortar and pestle previously cooled with liquid nitrogen. Chromosomal DNA was extracted from the obtained pulverized cells using DNeasy Plant Mini Kit (Qiagen).

(1-2) Preparation of Plasmid Containing AstrpC Disruption Cassette Plasmid pUC19, upstream region 1 for homologous recombination (As upstream region 1), pyrG gene which is a transformation marker that complements uridine / uracil requirement, and A plasmid for construction in which the downstream region 1 (As downstream region 1) for homologous recombination was sequentially ligated was prepared as follows.

In order to amplify the As upstream region 1, AspyrG gene and As downstream region 1, the chromosomal DNA of Aspergillus soja NBRC4239 obtained in the above (1-1) as a template DNA, and Q5 Hot Start High-Fidelity as a PCR enzyme Using 2X Master Mix (New England Biolabs) and T100 thermal cycler (Bio-Rad) as the device, PCR was performed according to the protocol attached to the enzyme.

It is the primer of primer sequence number 1-6 used in order to amplify As upstream region 1, AspyrG gene, and As downstream region 1. The amplified DNA fragment was purified using QIAquick PCR Purification Kit (Qiagen).

As pUC19, pUC19 linearized Vector attached to In-Fusion HD Cloning Kit (Clontech) was used. Using the In-Fusion Cloning Site at the multi-cloning site of pUC19, the three DNA fragments amplified were ligated using the In-Fusion HD Cloning Kit described above according to the protocol attached to the kit, and used for construction. A plasmid was obtained.

With the resulting plasmid for construct, ECOS Competent E., which is a competent cell. E. coli JM109 (Nippon Gene) was transformed according to the manufacturer's instructions to obtain transformed E. coli.

The obtained transformed E. coli was cultured overnight at 37 ° C. in an LB liquid medium containing 50 μg / ml ampicillin. The culture solution after the culture was centrifuged to recover the cells. About the obtained microbial cell, plasmid DNA was extracted according to the protocol attached to the kit using FastGene Plasmid Mini Kit (Nippon Genetics).

A plasmid was obtained in which the sequence of As upstream region 1-Aspir G-As downstream region 1 was linked in order to the multicloning site of pUC19. The obtained plasmid was designated as pTrpC_HR.

(1-3) Using the plasmid pTrpC_HR obtained in (1-2) above as the amplification template DNA for the AstrpC disruption cassette, PCR and purification of the PCR product are performed according to the description in (1-2) above. As a result, a cassette for destroying AstrpC was obtained. The used primer is a primer of sequence number 7-8.

As described above, an AsrpC destruction cassette in which the sequence of the As upstream region 1-AspyrG-As downstream region 1 was sequentially connected was obtained.

(1-4) Improvement of the pyrG gene and gene targeting efficiency on the chromosome of Aspergillus soja NBRC4239 strain as a transformed host of Aspergillus soja KP-del strain according to the description in Example 2 of Japanese Patent No. 6261039 Therefore, an Aspergillus soja KP-del strain in which the ku70 gene, which is a gene involved in non-homologous recombination, was disrupted was prepared. FIG. 2 shows an outline of transformation of Aspergillus soja KP-del strain using the AstrpC disruption cassette. As shown in FIG. 2, by transforming with the cassette for disrupting AstrpC, the pyrG gene is introduced into the locus of trpC gene on the chromosome of Aspergillus soja KP-del strain, and trpC gene is deficient. And an AstrpC disruption strain having a pyrG gene was prepared.

Inoculate 100 ml of polypeptone dextrin liquid medium containing 20 mM uracil and 20 mM uridine into a 500 ml Erlenmeyer flask and inoculate the conidia of Aspergillus sojae KP-del strain, and after shaking culture at 30 ° C. for about 20 hours, the cells Was recovered. Protoplasts were prepared from the collected cells. The obtained protoplast and 20 μg of AstrpC disruption cassette were used for transformation by protoplast PEG method, and then Czapek-Dox minimal medium containing 1 mM tryptophan, 0.5% (w / v) agar and 1.2 M sorbitol (Difco; pH 6) was used, and incubated at 30 ° C. for 5 days or longer to obtain transformed Aspergillus soya as having colony-forming ability.

In the transformed Aspergillus sojae in which AsrpC is disrupted, the host's uracil requirement is complemented by the AspyrG gene inserted at the AstrpC gene position, and instead it becomes tryptophan requirement.

(1-5) Selection of AsstrC-disrupted strain A strain in which the AsstrC gene was disrupted was selected by confirming by PCR using the chromosomal DNA of transformed Aspergillus soja extracted according to (1-2) above as a template. . The used primer is a primer of sequence number 9-12.

By performing the above PCR, it was confirmed by agarose electrophoresis that a 692 bp PCR product derived from the AsstrC gene was not generated, and a 439 bp PCR product derived from the Aslig1 gene that was not to be disrupted was generated, and an AsrpC disrupted strain was selected. did.

The selected AstrC disruption strain was subjected to PCR and restriction enzyme digestion treatment of the PCR product to confirm that the AsrpC disruption cassette was introduced by homologous recombination. The used primer is a primer of sequence number 13-14.

In the AstrpC disruption strain, a 5.7 kb PCR product was generated, and digestion with KpnI resulted in fragments of 2.4 kb, 2.1 kb and 1.2 kb. In the host, a 6.4 kb PCR product was generated and digested with KpnI to generate 3.5 kb and 2.9 kb fragments.

(1-6) Confirmation of Tryptophan Requirement AstrpC disruption strains confirmed to have been introduced by homologous recombination with the AsrpC disruption cassette were placed on plates of Czapek-Dox minimal agar medium containing or not containing 1 mM tryptophan, respectively. It was inoculated, incubated at 30 ° C. for 4 days, and confirmed to grow only in a medium containing tryptophan.

(1-7) Confirmation of AstrpC-disrupted strain for 5-FAA resistance AstrpC-disrupted strain and Aspergillus soja NBRC4239 strain as a control, potato dextrose agar medium containing 0.04% (w / v) 5-FAA and 1 mM tryptophan ( Nissui Pharmaceutical Co., Ltd.) was inoculated and incubated at 30 ° C. for 4 days. The NBRC4239 strain did not grow at all, while the AstrC disrupted strain grew. Thereby, it was confirmed that the AstrpC-disrupted strain showed resistance to 5-FAA. 5-FAA (5-fluoroanthranilic acid; Tokyo Chemical Industry Co., Ltd.) was used by preparing a 10% solution using ethanol and adding it to the medium so that the final concentration was the above concentration.

When the 5-FAA concentration was changed to 0.02% (w / v) in the same manner, both the AstrpC-disrupted strain and the NBRC4239 strain grew. Therefore, it was found that the AstrpC-disrupted strain can be selected by using 0.04% (w / v) 5-FAA against Aspergillus soya.

2. Gene disruption using AstrpC marker gene (2-1) As a gene to be disrupted, a poly predicted to be a region of 3347080-3344733 of scaffold00063 from the public genome database (BioProject Accession: PRJDA60265) of Aspergillus soja NBRC4239 strain (ADP-ribose) We decided to target the polymerase gene. This gene was designated as Asparp1 gene.

(2-2) Preparation of plasmid containing Asparp1 disruption cassette According to the method described in (1-2) above, plasmid pUC19 was subjected to upstream region 2 for homologous recombination (As upstream region 2), loop-out. Plasmid pPARP1_LO_Trp was prepared by linking region 1 for As (As loopout region 1), AstrpC gene and downstream region 2 for homologous recombination (As downstream region 2) in this order. The used primer is a primer of sequence number 15-22.

(2-3) Using the plasmid pPARP1_LO_Trp obtained in (2-2) above as the amplification template DNA for the Asparp1 disruption cassette, PCR and purification of the PCR product are performed according to the description in (1-3) above. As a result, an Asparp1 destruction cassette was obtained. The used primer is a primer of sequence number 23-24.

As described above, an Asparp1 disruption cassette was obtained in which the sequence of As upstream region 2-As loopout region 1-AstrpC-As downstream region 2 was sequentially linked.

(2-4) Production of Asparp1 Disrupted Strain An Asparp1 disrupted strain was prepared according to the following procedure. In addition, FIG. 3 shows an outline of transformation of an AstrpC disruption strain using an Asparp1 disruption cassette. As shown in FIG. 3, by transforming with an Asparp1 disruption cassette, the trpC gene is introduced into the locus of the parp1 gene on the chromosome of the AsrpC disruption strain, the parp1 gene is deleted, and A transformed Aspergillus sojae having the trpC gene was prepared.

The conidia of the AsrpC-disrupted strain were inoculated into 100 ml of a polypeptone dextrin liquid medium containing 1 mM tryptophan in a 500 ml Erlenmeyer flask, and cultured after shaking at 30 ° C. for about 24 hours, and then the cells were collected. Protoplasts were prepared from the collected cells. The obtained protoplast and 20 μg of Asparp1 disruption cassette were used for transformation by protoplast PEG method, and then using Czapek-Dox minimal medium containing 0.5% (w / v) agar and 1.2 M sorbitol. Incubated at 30 ° C. for 5 days or longer to obtain transformed Aspergillus soya as having colony-forming ability.

In transformed Aspergillus soya with Asparp1 disrupted, the host tryptophan requirement is complemented by the AsstrpC gene inserted at the Asparp1 gene position.

(2-5) Selection of Asparp1 Disrupted Strain According to the description in (1-5) above, a chromosomal DNA was extracted from the transformed strain and confirmed by PCR to select a strain in which the Asparp1 gene was disrupted. The used primer is a primer of sequence number 25-28.

By carrying out the above PCR, it was confirmed by agarose gel electrophoresis that a 460 bp PCR product derived from the Asparp1 gene was not generated and a 720 bp PCR product derived from the AsPtef promoter that was not subject to destruction was generated. The results of agarose gel electrophoresis are shown in FIG. 4A. As shown in FIG. 4A, in the transformants 2, 6, and 7, no PCR product (PARP) of the parp1 gene was generated.

The selected Asparp1 disruption strain was subjected to PCR to confirm that the Asparp1 disruption cassette was introduced by homologous recombination by agarose gel electrophoresis. The used primer is a primer of sequence number 29-30.

The results of agarose gel electrophoresis are shown in FIG. 4B. As shown in FIG. 4B, among the transformants 2, 6 and 7, transformants 2 and 7 produced a 9.6 kb PCR product. In the host, a 5.3 kb PCR product was generated. Thus, transformants 2 and 7 (PARP_2 and PARP_7, respectively) were used as Asparp1-disrupted strains.

3. Removal of AsrpC marker gene introduced (3-1) Confirmation of AsrpC-removed strain As shown schematically in FIG. 5, the trpC gene and the parp1 gene were removed by loop-out of the trpC gene introduced into the Asparp1-disrupted strain as follows The strains from which the was removed were selected.

Asparp1-disrupted conidia are smeared on a plate of potato dextrose agar (Nissui Pharmaceutical) containing 1 mM tryptophan and 0.04% (w / v) 5-FAA and incubated at 30 ° C. for 4 days. did. FIG. 5 shows the number of conidia sprayed PARP_2 and PARP_7, the number of resistant strains that appeared, and the frequency of resistant strains calculated from them.

Chromosomal DNA was extracted from the resulting 5-FAA-resistant strain and PCR was performed using the primers of SEQ ID NOs: 29 to 30 to determine that the strain was a AsrpC-removed strain in which loop-out occurred and AsrpC was removed. Confirmed by electrophoresis. FIG. 6A shows the results of agarose gel electrophoresis for seven 5-FAA resistant strains selected from the PARP_7 strain.

As FIG. 6A shows, a loop-out resulted in a 3.0 kb PCR product as expected. If loopout does not occur, a 9.6 kb PCR product is generated.

Also, by extracting chromosomal DNA from the AsrpC-removed strain and performing PCR using the primers of SEQ ID NOs: 9 to 12, a 692 bp PCR product derived from the AsstrC gene is not generated, and 439 bp derived from the Aslig1 gene that is not a target for destruction. The occurrence of the PCR product was confirmed by agarose electrophoresis. FIG. 6B shows the results of agarose gel electrophoresis for seven 5-FAA resistant strains selected from the PARP_7 strain. These seven strains were selected as AstrC-removed strains.

(3-2) Confirmation of Tryptophan Requirement By inoculating the selected AsrpC-removed strain on a plate of Czapek-Dox minimal agar medium with or without 1 mM tryptophan, and incubating at 30 ° C. for 4 days, It was confirmed that the AsrpC-removed strain grows only in a medium containing tryptophan, that is, it is tryptophan-requiring. It was also shown that AstrpC can be removed by using 5-FAA.

[Example 2. Use of the trpC gene as a selectable marker gene that can be used in the marker recycling method in Aspergillus oryzae]
1. In the same manner as in Production Example 1 of AotrpC-disrupted strain, using the chromosomal DNA of Aspergillus oryzae RIB40 strain as a template, upstream region 1 for homologous recombination (Ao upstream region 1) and downstream region 1 for homologous recombination ( After obtaining the DNA fragment of the Ao downstream region 1), a plasmid for construction pAoTrpC_HR was constructed in which the Ao upstream region 1, the AspyrG gene, and the Ao downstream region 1 were sequentially ligated to the plasmid pUC19. The used primer is a primer of sequence number 31-34.

Next, in the same manner as in Example 1, the AotrpC disruption cassette was amplified using the plasmid pAoTrpC_HR as the template DNA. The used primer is a primer of sequence number 35-36.

As described above, an AotrpC disruption cassette in which the sequences of the Ao upstream region 1-AspirrG-Ao downstream region 1 were sequentially connected was obtained.

As a host for producing an AotrpC-disrupted strain, Aspergillus oryzae RkuN16ptr1 strain (Mol. Genet. Genomics, 275: 460, 2006, Biosci. Biotechnol. Biochem, 70: 135, as described in Japanese Patent No. 5704609. , 2006). In the RkuN16ptr1 strain, the pyrG gene and the ku70 gene are disrupted.

A conidia of Aspergillus oryzae RkuN16ptr1 strain was inoculated into 100 ml of a polypeptone dextrin liquid medium containing 10 mM uracil in a 500 ml Erlenmeyer flask, and after shaking culture at 30 ° C. for about 20 hours, the cells were collected. Protoplasts were prepared from the collected cells. The obtained protoplast and 20 μg of AotrpC disruption cassette were used for transformation by the protoplast PEG method, and then Czapek-Dox minimal medium containing 1 mM tryptophan, 0.5% (w / v) agar and 1.2 M sorbitol Was used and incubated at 30 ° C. for 5 days or longer to obtain transformed Aspergillus oryzae having colony-forming ability.

In transformed Aspergillus oryzae in which AotrpC is disrupted, the host's uracil / uridine requirement is complemented by the AspyrG gene inserted at the position of the AotrpC gene, and instead it becomes tryptophan requirement.

Then, in the same manner as in Example 1, a strain in which the AotrpC gene was disrupted was selected by confirming by PCR using the chromosomal DNA of transformed Aspergillus oryzae as a template. The used primer is a primer of sequence number 9-10 and 37-38.

By carrying out the above PCR, it was confirmed by agarose electrophoresis that a 692 bp PCR product derived from the AotrpC gene was not generated and a 462 bp PCR product derived from the Aolig1 gene that was not subject to destruction was generated, and an AotrpC disrupted strain was selected. did.

The selected AotrpC disruption strain was subjected to PCR and restriction enzyme digestion of the PCR product to confirm that the AotrpC disruption cassette was introduced by homologous recombination. The primers used are those of SEQ ID NOs: 14 and 39.

In the AotrpC disruption strain, a 5.7 kb PCR product was generated, and digestion with KpnI resulted in 2.5 kb, 2.1 kb and 1.2 kb fragments. In the host, a 6.3 kb PCR product was produced, and digesting this with KpnI produced 3.5 kb and 2.8 kb fragments.

AotrpC disruption strains confirmed to have introduced the AotrpC disruption cassette by homologous recombination were inoculated on plates of Czapek-Dox minimal agar medium with or without 1 mM tryptophan at 30 ° C. for 5 days. It was incubated and confirmed to grow only in a medium containing tryptophan.

An AotrpC-disrupted strain and an Aspergillus oryzae RIB40 strain as a control were inoculated on a plate of Sabouraud dextrose agar medium (BD) containing 0.12% (w / v) 5-FAA and 1 mM tryptophan at 30 ° C. for 4 days. Incubated. The RIB40 strain did not grow at all, whereas the AotrpC disrupted strain grew. Thereby, it was confirmed that the AotrpC-disrupted strain showed resistance to 5-FAA.

When the 5-FAA concentration was changed to 0.1% (w / v) in the same manner, both the AotrpC disrupted strain and the RIB40 strain grew. Therefore, it was found that an AotrpC-disrupted strain can be selected by using 0.12% (w / v) 5-FAA against Aspergillus oryzae.

2. As a gene disruption target gene using the AotrpC marker gene, a predicted gene with unknown function in the region of 1456616-1457378 of SC102 from the public genome database (BioProject Accession: PRJNA20809) of Aspergillus oryzae RIB40 strain was used. This gene was designated as AohypG.

In the same manner as in Example 1, using the chromosomal DNA of Aspergillus oryzae RIB40 strain as a template, upstream region 2 for homologous recombination (Ao upstream region 2), AotrpC gene, region for loopout (Ao loopout region 1) ) And downstream region 2 (Ao downstream region 2) for homologous recombination, and then ligated to plasmid pUC19 in sequence Ao upstream region 2, AotrpC gene, Ao loopout region 1 and Ao downstream region 2 The constructed plasmid pAoHypG_LO_TrpC for construction was prepared. The used primer is a primer of sequence number 20 and 40-46.

In the same manner as in Example 1, using the plasmid pAoHypG_LO_TrpC obtained above as a template DNA, an AohypG disruption cassette was obtained. The used primer is a primer of sequence number 47-48.

As described above, an AohypG disruption cassette in which the sequences of the Ao upstream region 2-AotrpC-Ao loop-out region 1-Ao downstream region 2 were sequentially connected was obtained.

An AohypG disrupted strain was prepared by the following procedure. An outline of transformation of the AotrpC disruption strain using the AohypG disruption cassette is shown in FIG. As shown in FIG. 7, by transforming with an AohypG disruption cassette, the trpC gene is introduced into the locus of the hypG gene on the chromosome of the AotrpC disruption strain, the hypG gene is deleted, and A transformed Aspergillus oryzae having the trpC gene was prepared.

Inoculate 100 ml of potato dextrose liquid medium (BD) containing 1 mM tryptophan in a 500 ml Erlenmeyer flask with the conidia of Aspergillus oryzae AotrpC disrupted strain, and perform shaking culture at 30 ° C. for about 24 hours. Was recovered. Protoplasts were prepared from the collected cells. Using the obtained protoplast and 20 μg of AohypG disruption cassette, transformation was performed by protoplast PEG method, and then using Czapek-Dox minimal medium containing 0.5% (w / v) agar and 1.2 M sorbitol. Incubated at 30 ° C. for 5 days or longer to obtain transformed Aspergillus oryzae having colony-forming ability.

In transformed Aspergillus oryzae in which AohypG is disrupted, the host tryptophan requirement is complemented by the AotrpC gene inserted at the position of the AohypG gene.

In the same manner as in Example 1, chromosomal DNA was extracted from the transformed strain and confirmed by PCR to select a strain in which the AohypG gene was disrupted. The primers used are those of SEQ ID NOs: 37-38 and 49-50.

By carrying out the above PCR, it was confirmed by agarose electrophoresis that a 702 bp PCR product derived from the AohypG gene was not generated and a 462 bp PCR product derived from Aolig1 that was not the target of destruction was generated, and an AohypG disrupted strain was selected. .

The selected AohypG disruption strain was subjected to PCR to confirm that the AohypG disruption cassette was introduced by homologous recombination. The used primer is a primer of sequence number 51-52.

In the AohypG disruption strain, an 8.6 kb PCR product was generated. In the host, a 4.3 kb PCR product was generated.

3. Removal of AotrpC Marker Gene Introduced As schematically shown in FIG. 8, a strain from which trpC gene and hypG gene were removed from which trpC gene introduced into AohypG disrupted strain was removed by loop-out was selected by the following procedure.

The conidia of the AohypG-disrupted strain were smeared on a plate of Sabouraud dextrose agar medium (BD) containing 1 mM tryptophan and 0.12% (w / v) 5-FAA, and incubated at 30 ° C. for 5 days or more.

Chromosomal DNA is extracted from the resulting 5-FAA-resistant strain and PCR is performed using the primers of SEQ ID NOs: 51 to 52, so that it is determined that the AotrpC-removed strain has a loopout and AotrpC has been removed. Confirmed by electrophoresis. FIG. 9A shows the results of agarose gel electrophoresis for the resulting 5-FAA resistant strain.

As shown in FIG. 9A, in any 5-FAA resistant strain, a loop-out occurred to produce a 2.7 kb product as expected. If loopout does not occur, an 8.6 kb PCR product is generated.

Also, by extracting chromosomal DNA from a 5-FAA resistant strain and performing PCR using the primers of SEQ ID NOs: 9 to 10 and 37 to 38, a 692 bp PCR product derived from the AotrpC gene does not occur, and It was confirmed by agarose electrophoresis that a 462 bp PCR product derived from a non-Aolig1 gene was generated. The results of agarose gel electrophoresis for 6 5-FAA resistant strains are shown in FIG. 9B. These 6 strains were selected as AotrpC-removed strains.

In addition, it was confirmed by the same method as in Example 1 that the AotrpC-removed strain grows only in a medium containing tryptophan, that is, is tryptophan-requiring. Moreover, it was shown that AotrpC can be removed by using 5-FAA.

[Example 3. Use of the trpC gene as a selectable marker gene that can be used in the marker recycling method in Aspergillus niger]
1. In the same manner as in Production Example 1 of AntrpC-disrupted strain, upstream region 1 (An upstream region) for homologous recombination using chromosomal DNA of Aspergillus niger A1179 strain (ΔkusA, pyrG-; obtained from Fungal Genetics Stock Center) as a template 1) and a plasmid for construct in which a DNA fragment of downstream region 1 (An downstream region 1) for homologous recombination is obtained, and then an upstream region 1, AspyrG gene, and An downstream region 1 are sequentially ligated to plasmid pUC19. pAnTrpC_HR was prepared. The used primer is a primer of sequence number 53-56.

Then, in the same manner as in Example 1, the AntrpC disruption cassette was amplified using the plasmid pAnTrpC_HR as the template DNA. The used primer is a primer of sequence number 57-58.

As described above, an AntrpC disruption cassette in which the sequences of the An upstream region 1-AspirG-An downstream region 1 were sequentially connected was obtained.

In a 500 ml Erlenmeyer flask, 100 ml of potato dextrose liquid medium (BD) containing 10 mM uracil and 10 mM uridine was inoculated with conidia of Aspergillus niger A1179 strain, and cultured with shaking at 30 ° C. for about 16 hours. Thereafter, the cells were collected. Protoplasts were prepared from the collected cells. The resulting protoplast and 20 μg of AntrpC disruption cassette were used for transformation by protoplast PEG method, and then Czapek-Dox minimal medium containing 1 mM tryptophan, 0.5% (w / v) agar and 1.2 M sorbitol Was used and incubated at 30 ° C. for 4 days or longer to obtain transformed Aspergillus niger as having colony-forming ability.

In transformed Aspergillus niger in which AntrpC is disrupted, the host's uracil requirement is complemented by the AspyrG gene inserted at the location of the AntrpC gene, and instead it becomes tryptophan requirement.

Then, in the same manner as in Example 1, a strain in which the AntrpC gene was disrupted was selected by confirming by PCR using the chromosomal DNA of transformed Aspergillus niger as a template. The used primer is a primer of sequence number 59-62.

By performing the above PCR, it was confirmed by agarose electrophoresis that a 420 bp PCR product derived from the AntrpC gene was not generated, and a 862 bp PCR product derived from the introduced AspyrG gene was generated, and an AntrpC disruption strain was selected. .

The selected AntrpC-disrupted strain was subjected to PCR and restriction enzyme digestion of the PCR product to confirm that the AntrpC disruption cassette was introduced by homologous recombination. The used primer is a primer of sequence number 63-64.

In the AntrpC-disrupted strain, a 5.5 kb PCR product was generated, which was digested with KpnI to generate 2.4 kb, 2.0 kb and 1.2 kb fragments. In the host, a 6.1 kb PCR product was produced, but the PCR product was not digested with KpnI.

The AntrpC disruption strains confirmed to have introduced the AntrpC disruption cassette by homologous recombination were inoculated on plates of Czapek-Dox minimal agar medium with or without 1 mM tryptophan, respectively, at 30 ° C. for 7 days. It was incubated and confirmed to grow only in a medium containing tryptophan.

2. As a gene disruption disruption gene using the AntrpC marker gene, the gene of TpsC, which is trehalose 6-phosphate synthase possessed by Aspergillus niger (BMC Microbiol 14, 90 (2014), [Website] https: //www.ncbi. nlm.nih.gov/pmc/articles/PMC3991884/). The gene was named AntpsC.

In the same manner as in Example 1, using the chromosomal DNA of Aspergillus niger strain A1179 as a template, upstream region 2 for homologous recombination (An upstream region 2), region for loopout (An loopout region 1), AntrpC After obtaining the DNA fragment of the gene and the downstream region 2 (An downstream region 2) for homologous recombination, the An upstream region 2, the An loopout region 1, the AntrpC gene, and the An downstream region 2 are sequentially ligated to the plasmid pUC19. The constructed plasmid pAnTpsC_LO_TrpC for construction was prepared. The used primer is a primer of sequence number 65-72.

In the same manner as in Example 1, an AntpsC disruption cassette was obtained using the plasmid pAnTpsC_LO_TrpC obtained above as the template DNA. The used primer is a primer of sequence number 73-74.

As described above, an AntpsC destruction cassette in which the sequences of the An upstream region 2-An loop-out region 1-AntrpC-An downstream region 2 were sequentially connected was obtained.

The AntpsC disruption strain was produced by transforming the AntrpC disruption strain using the AntpsC disruption cassette by the following procedure.

Inoculate 100 ml of potato dextrose liquid medium containing 1 mM tryptophan in a 500 ml Erlenmeyer flask with conidia of the Aspergillus niger AntrpC disrupted strain, and after shaking culture at 30 ° C. for about 16 hours, collect the cells. did. Protoplasts were prepared from the collected cells. The obtained protoplast and 20 μg of AntpsC disruption cassette were used for transformation by protoplast PEG method, and then using Czapek-Dox minimal medium containing 0.5% (w / v) agar and 1.2 M sorbitol. Incubated at 30 ° C. for 4 days or longer to obtain transformed Aspergillus niger as having colony-forming ability.

In transformed Aspergillus niger in which AntpsC is disrupted, the host tryptophan requirement is complemented by the AntrpC gene inserted at the position of the AntpsC gene.

In the same manner as in Example 1, chromosomal DNA was extracted from the transformed strain and confirmed by PCR to select a strain in which the AntpsC gene was disrupted. The primers used are those of SEQ ID NOs: 61-62 and 75-76.

By carrying out the above PCR, it was confirmed by agarose electrophoresis that a 430 bp PCR product derived from the AntpsC gene was not generated and a 862 bp PCR product derived from the introduced AspyrG gene was generated, and an AntpsC disruption strain was selected.

The selected AntpsC disruption strain was subjected to PCR to confirm that the AntpsC disruption cassette was introduced by homologous recombination. The used primer is a primer of sequence number 77-78.

The AntpsC disruption strain produced a 9.1 kb PCR product. In the host, a 5.9 kb PCR product was generated.

3. Removal of introduced AntrpC marker gene Conidia of AntpsC-disrupted strain are spread on a plate of potato dextrose agar containing 1 mM tryptophan and 0.015% (w / v) 5-FAA, and incubated at 30 ° C. for 4 days or more did.

Chromosomal DNA was extracted from the resulting 5-FAA-resistant strain and PCR was performed using the primers of SEQ ID NOs: 77 to 78 to confirm that it was an AntrpC-removed strain in which loopout occurred and AntrpC was removed.

The loop-out produced a 2.9 kb product as expected. If no loop out occurs, a 9.1 kb PCR product is generated.

Further, by the same method as in Example 1, it was confirmed that the AntrpC-removed strain grows only on a medium containing tryptophan, that is, is tryptophan-requiring. It was also shown that AntrpC can be removed by using 5-FAA.

[Example 4. Preparation of double disruption strain deficient in AspyrG and AsstrC genes]
According to the following procedure, the AspyrG / AstrpC double-disrupted strain was prepared by disrupting the AsstrG gene in the AspyrG-disrupted strain of Aspergillus soja with the AspyrG gene and then removing the introduced AspyrG by loop-out. FIG. 10 shows an outline of the procedure for producing the AspyrG and AsrpC double disruption strain.

1. Preparation of cassette for destroying AstrpC capable of looping out Inverse PCR and purification of the PCR product were performed using the plasmid pTrpC_HR prepared in Example 1 as a template to obtain a vector fragment. The used primer is a primer of sequence number 79-80.

Using the chromosomal DNA of Aspergillus soja NBRC4239 strain as a template, PCR and purification of the PCR product were performed to obtain region 2 for loop-out (As loop-out region 2). The used primer is a primer of sequence number 81-82.

The obtained vector fragment and the As loopout region 2 were used for ligation according to the method described in Example 1-2 (1-2), and the As upstream region 1-As loopout region 2 was added to the multicloning site of pUC19. -A plasmid in which the sequences of AspyrG-As downstream region 1 were sequentially ligated was obtained. The obtained plasmid was designated as pTrpC_LO_pyrG.

Next, in the same manner as in Example 1 (1-3), a plasmid for disrupting AstrpC that can be looped out was amplified using the plasmid plasmid pTrpC_LO_pyrG as the template DNA. The used primer is a primer of sequence number 7-8.

As described above, an AsrpC disruption cassette capable of looping out was obtained in which the sequence of the As upstream region 1 -As loopout region 2 -AspyrG-As downstream region 1 was sequentially linked.

2. According to the method described in (1-4) of Preparation Example 1 of AsstrC-disrupted strain, transformed Aspergillus soya KP-del strain is transformed with an AsrpC-disrupted cassette capable of loop-out, thereby transforming Aspergillus・ I got a soya.

In transformed Aspergillus soya with AsstrC disrupted, the host's uracil / uridine requirement is complemented by the AspyrG gene inserted at the AstrpC gene position, but instead becomes tryptophan requirement.

Conidia were collected from the plate on which transformed Aspergillus soya was produced using 0.01% (w / v) Tween 80 solution. The conidia suspension appropriately diluted with the same solution was smeared on a plate of potato dextrose agar medium containing 0.04% (w / v) 5-FAA and 1 mM tryptophan, and incubated at 30 ° C. for 4 days.

The resulting 5-FAA resistant strains were each inoculated into a plate of Czapek-Dox minimal agar medium with or without 1 mM tryptophan, incubated at 30 ° C. for 4 days, and grown only on medium containing tryptophan, That is, a strain showing tryptophan requirement was selected.

Extraction of chromosomal DNA from a tryptophan-requiring strain and PCR and restriction enzyme digestion of the PCR product reveals that an AstrC disruption cassette capable of looping out has been introduced by homologous recombination by agarose gel electrophoresis. confirmed. The primers used are those of SEQ ID NOs: 13 and 83.

In the AstrpC disruption strain, an 8.8 kb PCR product was generated, which was not cleaved when digested with BamHI. In the host, an 8.2 kb PCR product was produced, and digestion with BamHI produced 4.3 kb and 3.8 kb fragments.

3. Preparation of AspyrG / AstrpC double disruption strain Conidia of the AsstrC disruption strain obtained above were placed on a plate of Czapek-Dox minimal agar medium containing 1 mM tryptophan, 20 mM uracil and 0.3% (w / v) 5-FOA. The 5-FOA resistant strain was selected by smearing and incubating at 30 ° C. for 5 days or longer. In addition, 5-FOA (5-fluoroorotic acid; Fluorochem) is prepared by adding a 1.2% aqueous solution to pH 6 and then filter sterilizing it to the medium so that the final concentration is the above concentration. used.

By extracting chromosomal DNA from the 5-FOA resistant strain and performing PCR using the primers of SEQ ID NOs: 13 and 83, the 5-FOA resistant strain can be further isolated from the AsrpC-disrupted strain by the loop-out. Was confirmed to be an AspyrG / AstropC double disrupted strain. That is, it was confirmed that the PCR produced a 3.9 kb PCR product as expected after the loop-out. As described above, the PCR product obtained before looping out is 8.8 kb.

4). Confirmation of Tryptophan Requirement and Uracil Requirement AspyrG / AstrpC double disruption strains were inoculated on 4 types of agar plates shown in (1) to (4) below and incubated at 30 ° C. for 5 days or more.
(1) Czapek-Dox minimal agar medium (2) Czapek-Dox minimal agar medium containing 1 mM tryptophan (3) Czapek-Dox minimal agar medium containing 20 mM uracil (4) Czapek-Dox agar medium containing 1 mM tryptophan and 20 mM uracil Culture medium

It was confirmed that the AspyrG / AstrpC double disruption strain grew only in the medium containing the tryptophan and uracil of the above (4) and showed requirements for both tryptophan and uracil.

5. Confirmation of 5-FOA resistance and 5-FAA resistance Non-destructive strain (Aspergillus soya NBRC4239 strain), AspyrG disruption strain (Aspergillus soja KP-del strain), AstrpC disruption strain (produced in Example 1) and AspyrG. AsrppC The double-disrupted strains were used to evaluate the 5-FOA resistance and 5-FAA resistance of these strains by the following procedure.

A plate of potato dextrose agar medium containing 1 mM tryptophan, 20 mM uracil and 5-FOA and 5-FAA at the concentrations shown in Table 3 was inoculated and incubated at 30 ° C. for 4 days. The 5-FOA resistance and 5-FAA resistance of each strain were confirmed by the presence or absence of colony formation on the plate.

“+”: Growing, “−”: not growing

AspyrG disrupted and AsstrC disrupted strains could not grow due to the presence of 5-FAA and 5-FOA in the medium, respectively.

In Example 1, 0.04% (w / v) 5-FAA was used for selection of the ΔtrpC strain. In Example 4, 0.3% (w / v) 5-FOA was used for selection of the ΔpyrG strain. However, as Table 3 shows, any combination of these drugs used alone (0.3% (w / v) 5-FOA and 0.04% (w / v) 5-FAA) The strain did not grow.

On the other hand, when both drug concentrations were lowered to ½ and ¼, only the AspyrG / AstrpC double disruption strain grew. Since the effect was exhibited even when the concentration was lowered to 1/4, it was revealed that 5-FOA and 5-FAA are synergistically effective. It has been found that the use of such a medium makes it possible to select an AspyrG / AstrpC double disruption strain.

[Example 5. Simultaneous use of pyrG gene and trpC gene as selectable marker genes that can be used in the marker recycling method]
1. Production of Asnph Destruction Cassette As a target gene for disruption, it was decided to target a gene of protease predicted in the region of 1904082-1905244 of scaffold00003 from the public genome database (BioProject Accession: PRJDA60265) of Aspergillus soja NBRC4239 strain. This gene was designated as Asnph gene.

In accordance with the method described in Example 1 (1-2), plasmid pUC19 was subjected to upstream region 2 for homologous recombination (As upstream region 3) and region 3 for loopout (As loopout region 3). A plasmid pNpH_LO_pyrG was prepared by sequentially linking the AspyrG gene and the downstream region 3 for homologous recombination (As downstream region 3). The used primer is a primer of sequence number 84-89. The DNA fragment of AspyrG gene was the same as that prepared in (1-2) of Example 1.

Using the obtained plasmid pNpH_LO_pyrG as a template DNA, PCR and PCR product purification were performed according to the description in (1-3) above to obtain an Asnph destruction cassette. The used primer is a primer of sequence number 90-91.

As described above, an Asnph destruction cassette in which the sequence of the As upstream region 3-As loop-out region 3-AspyrG-As downstream region 3 was sequentially linked was obtained.

2. Production of Asparp1 / Asnph double disruption strain Asparp1 / Asnph double disruption strain by Asparp1 / Asnph double disruption strain transformation using Asparp1 disruption cassette and Asnph disruption cassette FIG. 11 shows an outline of the procedure up to the production of an AspyrG · AstropC double-removed strain obtained by looping out the nuclease.

In a 500 ml Erlenmeyer flask, 100 ml of a potato dextrose liquid medium containing 1 mM tryptophan, 20 mM uracil and 20 mM uridine was inoculated with conidia of the Aspergillus soya Aspyr G AsstrC double disruption strain prepared in Example 4, and about 24 at 30 ° C. After culturing with shaking, the cells were collected. Protoplasts were prepared from the collected cells. The obtained protoplast and 20 μg of Asparp1 disruption cassette (prepared in Example 1) and 20 μg of Asnph disruption cassette were transformed by protoplast PEG method, and then 0.5% (w / v) agar And Czapek-Dox minimal medium containing 1.2 M sorbitol was incubated at 30 ° C. for 5 days or longer to obtain transformed Aspergillus soya as having colony-forming ability.

The uracil requirement and tryptophan requirement of the host AspyrG / AstrpC double disruption strain are complemented by the introduced AspyrG gene and AsstrpC gene, respectively. Note that a strain in which only one of them is complemented cannot grow on the above medium.

In accordance with the description in (1-5) above, a chromosomal DNA was extracted from the transformed strain and confirmed by PCR to select a strain in which both Asparp1 gene and Asnph gene were disrupted. The primers used are those of SEQ ID NOs: 25-28 and 92-93.

By performing the above PCR, a 460 bp PCR product derived from Asparp1 gene and a 291 bp PCR product derived from Asnph were not generated, and a 720 bp PCR product derived from the AsPtef promoter that was not subject to destruction was generated by agarose electrophoresis. confirmed. The results of agarose gel electrophoresis are shown in FIG. The transformants 7, 9, and 13 shown in FIG. 12 were selected as Asparp1 · Asnph double disruption strains.

The selected Asparp1 / Asnph double disruption strain was confirmed by PCR to confirm that both the Asparp1 disruption cassette and Asnph disruption cassette were introduced by homologous recombination. The primers used are those of SEQ ID NOs: 29-30 and 94-95.

The Aspart1 gene was disrupted, resulting in a 9.6 kb PCR product. In the host, a 5.3 kb PCR product was generated. In addition, the Asnph gene was disrupted, resulting in an 8.5 kb PCR product. A 5.9 kb PCR product was generated in the host.

3. Removal of introduced AspyrG marker gene and AstrpC marker gene (3-1) Conidia of simultaneous loop-out Asparp1 / Asnph double disruption strain were obtained by using 1 mM tryptophan, 20 mM uracil, 0.08% (w / v) 5-FOA and A plate of potato dextrose agar medium containing 0.01% (w / v) 5-FAA was smeared and incubated at 30 ° C. for 10 days or longer.

The resulting 5-FOA-resistant and 5-FAA-resistant strains were inoculated on an agar plate of the same composition and confirmed to grow by incubating at 30 ° C. for 4 days.

Chromosomal DNA is extracted from the strains whose growth has been confirmed, and PCR is performed using the primers of SEQ ID NOs: 29-30, 94-95, 9-10, 11-12, and 61-62, whereby AspyrG marker gene and AsrpC The strain from which the marker gene was removed was confirmed by agarose gel electrophoresis. 13A to 13B show the results of agarose gel electrophoresis for the three strains after loop-out of the transformant 13 selected as the Asparp1 / Asnph double disruption strain in 2 above.

As shown in FIG. 13A, a loop-out occurred in the vicinity of the region where the Asparp1 gene originally existed, resulting in a 3.0 kb product as expected. If loopout does not occur, a 9.6 kb PCR product is generated.

As FIG. 13B shows, a loop-out occurred in the vicinity of the region where the Asnph gene originally existed, resulting in a 3.4 kb product as expected. If loopout does not occur, an 8.5 kb PCR product is generated.

It was confirmed by agarose electrophoresis that a 862 bp PCR product derived from the AspyrG gene and a 692 bp PCR product derived from the AsstrC gene were not generated, and a 439 bp PCR product derived from the Aslig1 gene that was not subject to destruction was generated. The results are shown in FIG. 13C. From these results, 3 strains after the loop-out of the transformant 13 were selected as AspyrG and AsrpC double-removed strains.

The AspyrG / AstrpC double-removed strain was inoculated on 4 types of agar plates shown in (1) to (4) below, and incubated at 30 ° C. for 5 days or longer.
(1) Czapek-Dox minimal agar medium (2) Czapek-Dox minimal agar medium containing 1 mM tryptophan (3) Czapek-Dox minimal agar medium containing 20 mM uracil (4) Czapek-Dox agar medium containing 1 mM tryptophan and 20 mM uracil Culture medium

The AspyrG / AstrpC double-removed strain grew only in the medium containing tryptophan and uracil of (4) above, and was confirmed to be required for both tryptophan and uracil. Thereby, it was confirmed that the two types of marker genes could be looped out simultaneously.

(3-2) Conidia of the two-stage loop-out Asparp1 / Asnph double disruption strain are spread on a plate of Czapek-Dox minimal agar medium containing 20 mM uracil and 0.3% (w / v) 5-FOA. Incubated at 30 ° C. for 5 days or longer. Conidia are collected from a plate on which a 5-FOA resistant strain (AspyrG-removed strain) has been generated using a 0.01% (w / v) Tween 80 solution, and appropriately diluted with the same solution, whereby a conidia suspension is obtained. A liquid was prepared.

An appropriate amount of the resulting conidial suspension was added to a potato dextrose agar medium containing 1 mM tryptophan, 20 mM uracil, 0.08% (w / v) 5-FOA and 0.01% (w / v) 5-FAA. The plate was smeared and incubated at 30 ° C. for 4 days.

The resulting 5-FOA / 5-FAA resistant strain was inoculated on a plate of an agar medium having the same composition and confirmed to grow by incubating at 30 ° C. for 4 days.

Chromosomal DNA was extracted from the strains that had been confirmed to grow, and PCR was performed by the method described in Example 3 (3-1) to remove the AspyrG marker gene and the AstrpC marker gene. It was confirmed that it was a removed strain.

In addition, it was confirmed by the method described in Example 3 (3-1) that the AspyrG · AstrpC double-removed strain showed requirements for both uracil and tryptophan. As a result, it was confirmed that the two-stage loop-out of the two kinds of marker genes was completed.

The transformed Aspergillus microorganism, composition and method which are one embodiment of the present invention can efficiently carry out transformation of an Aspergillus microorganism having high utility value in the food industry, and further, the structure or function of the Aspergillus microorganism It is possible to quickly detect new phenotypes due to simultaneous disruption of genes that are similar or related.

Claims (8)

Selection marker genes available to at least two markers Recycling Law on the chromosome deficient, a transformed Aspergillus (Aspergillus) microorganisms, said selection marker gene viewed including the trpC gene and pyrG genes, and host The transformed Aspergillus microorganism , wherein the organism is an Aspergillus microorganism other than Aspergillus acleutus . A composition for transforming Aspergillus microorganisms other than Aspergillus aculeatus , comprising at least two kinds of nucleic acid fragments comprising a loopout region and a selectable marker gene usable for marker recycling method between homologous recombination regions The nucleic acid fragment includes the nucleic acid fragment in which the selection marker gene is a trpC gene and the nucleic acid fragment in which the selection marker gene is a pyrG gene. The microorganisms belonging to the genus Aspergillus have host organisms of Aspergillus sojae, Aspergillus oryzae, Aspergillus tamarii, Aspergillus tamariis, Or the Aspergillus saitoi , the transformed Aspergillus microorganism or composition according to any one of claims 1-2. A transformed Aspergillus deficient in the first selectable marker gene usable in the marker recycling method on the chromosome and the second selectable marker gene usable in the marker recycling method, comprising the following steps (1) to (3): a method of manufacturing a microorganism, selectable marker gene of the first selection marker gene and said second, either is trpC gene and the other Ri Ah at pyrG gene, nutritional corresponding to trpC gene The substance is tryptophan, the tryptophan analog is 5-FAA, the nutrient corresponding to the pyrG gene is uracil and / or uridine, the uracil and / or uridine analog is 5-FOA, and the host Aspergillus acculeatus or higher Which is the genus Aspergillus microorganisms, said method:
(1) Using a nucleic acid fragment containing a loop-out region and a first selectable marker gene between a homologous recombination region and a transformed Aspergillus microorganism lacking the first selectable marker gene on the chromosome, Obtaining a transformed Aspergillus microorganism by homologous recombination using the second selectable marker gene as a target;
(2) By culturing the transformed Aspergillus microorganism obtained in the step (1) in the presence of a nutrient substance corresponding to the second selection marker gene, the first selection marker on a chromosome Selecting a transformed Aspergillus microorganism in which the gene is inserted and the second selectable marker gene on the chromosome is deleted; and
(3) The transformed Aspergillus microorganism selected in the step (2), the nutrient substance corresponding to the first selectable marker gene, the analog of the nutrient substance, and the nutrient corresponding to the second selectable marker gene A step of selecting a transformed Aspergillus microorganism lacking the first selection marker gene and the second selection marker gene on a chromosome by culturing in the presence of a sex substance. A transformed Aspergillus genus microorganism using the first selectable marker gene usable in the marker recycling method and the second selectable marker gene usable in the marker recycling method, comprising the following steps (A) to (B): a deficiency method of the two on the chromosome of a target gene, a selection marker gene of the first selection marker gene and the second is either one trpC gene and the other Ri Ah at pyrG gene, The trophic substance corresponding to the trpC gene is tryptophan, the tryptophan analog is 5-FAA, the trophic substance corresponding to the pyrG gene is uracil and / or uridine, and the uracil and / or uridine analog is 5- FOA and the host organism is Aspergillus culeatus) is a genus Aspergillus microorganisms other than, said method:
(A) A transformed Aspergillus microorganism lacking the first selectable marker gene and the second selectable marker gene on the chromosome, the loop-out region and the first selection between the homologous recombination regions for the first target gene A first nucleic acid fragment comprising a marker gene and a second nucleic acid fragment comprising a loop-out region and a second selectable marker gene between a first nucleic acid fragment comprising a marker gene and a homologous recombination region for a second target gene; And obtaining a transformed Aspergillus microorganism by homologous recombination using the second target gene as a target; and (B) transforming Aspergillus microorganism obtained in the step (A) with the first Staining by culturing in the absence of the nutrient substance corresponding to the selectable marker gene and the nutrient substance corresponding to the second selectable marker gene The first selection marker gene and said second selection marker gene is the step of selecting transformed Aspergillus microorganism inserted above. The method according to claim 5 , further comprising the following step (C):
(C) The transformed Aspergillus microorganism selected in the step (B) corresponds to the nutrient substance corresponding to the first selectable marker gene, the analog of the nutrient substance, and the second selectable marker gene By culturing in the presence of a nutrient substance and an analogue of the nutrient substance, the first selectable marker gene, the second selectable marker gene, the first target gene, and the second target on a chromosome A step of selecting a transformed Aspergillus microorganism lacking the gene. The concentration of the previous Symbol 5 -FAA is 0.005% (w / v) ~0.02 % (w / v), and,
Concentration before Symbol 5 -FOA is 0.05% (w / v) ~0.15 % (w / v), The method of claim 6. The microorganisms belonging to the genus Aspergillus have host organisms of Aspergillus sojae, Aspergillus oryzae, Aspergillus tamarii, Aspergillus tamariis, ) Or Aspergillus saitoi. 8. The method according to any one of claims 4 to 7 .